Single-stranded AAV Research Articles

In-Depth Comparison of Adeno-Associated Virus Containing Fractions after CsCl Ultracentrifugation Gradient Separation.

Recombinant adeno-associated viruses (rAAVs) play a pivotal role in the treatment of genetic diseases. However, current production and purification processes yield AAV-based preparations that often contain unwanted empty, partially filled or damaged viral particles and impurities, including residual host cell DNA and proteins, plasmid DNA, and viral aggregates. To precisely understand the composition of AAV preparations, we systematically compared four different single-stranded AAV (ssAAV) and self-complementary (scAAV) fractions extracted from the CsCl ultracentrifugation gradient using established methods (transduction efficiency, analytical ultracentrifugation (AUC), quantitative and digital droplet PCR (qPCR and ddPCR), transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA)) alongside newer techniques (multiplex ddPCR, multi-angle light-scattering coupled to size-exclusion chromatography (SEC-MALS), multi-angle dynamic light scattering (MADLS), and high-throughput sequencing (HTS)). Suboptimal particle separation within the fractions resulted in unexpectedly similar infectivity levels. No single technique could simultaneously provide comprehensive insights in the presence of both bioactive particles and contaminants. Notably, multiplex ddPCR revealed distinct vector genome fragmentation patterns, differing between ssAAV and scAAV. This highlights the urgent need for innovative analytical and production approaches to optimize AAV vector production and enhance therapeutic outcomes.

Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to ½ binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (i) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (ii) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (iii) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (iv) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo, and (v) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE ½ binding site, and insertion of with a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y (GenY) AAV vectors, which are serotype-, transgene-, or promoter-agnostic, should be useful in human gene therapy.

A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging.

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate.

Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to crude lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.

Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.

Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses.

We have described the development of capsid-modified next-generation AAV vectors for both AAV2 and AAV3 serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses. We have also described the development of genome-modified AAV vectors, in which the transcriptionally inactive, single-stranded AAV genome was modified to achieve improved transgene expression. Here, we describe that combination of capsid modifications and genome modifications leads to the generation of optimized AAV serotype vectors, which transduce cells and tissues more efficiently, both in vitro and in vivo, at ∼20-30-fold reduced doses. These studies have significant implications in the potential use of the optimized AAV serotype vectors in human gene therapy.

91. Development of Optimized [“Opt”] AAV Vectors by Combining Capsid-Modified NextGen and Genome-Modified GenX AAV Vectors for High-Efficiency Transduction at Further Reduced Doses

There is little doubt that AAV did not evolve for the purposes of delivery of therapeutic genes. The use of first generation AAV vectors, albeit successful, is unlikely to reach its full potential. We have described the development of capsid-modified next generation [NextGen] AAV vectors for both AAV2 (Proc Natl Acad Sci USA, 105: 7827-7832, 2008; Mol Ther., 18: 2048-2056, 2010; Vaccine, 30: 3908-3917, 2012; PLoS One, 8: e59142, 2013) and AAV3 (Gene Ther., 19: 375-384, 2012; Hum Gene Ther., 25: 1023-1034, 2014) serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses Fig. 1AFig. 1A). We have also described the development of genome-modified generation X [GenX] AAV vectors (J Virol., 89: 952-961, 2015), in which the transcriptionally-inactive, single-stranded AAV genome was modified to achieve improved transgene expression (Fig. 1BFig. 1B). Thus, we reasoned that encapsidation of GenX AAV genomes into NextGen AAV capsid might lead to further increased transduction at further reduced vector doses. To this end, the following sets of ssAAV2 as well as ssAAV3 serotype vectors containing the firefly luciferase (Fluc) reporter gene were generated: (i) wild-type (WT) genome and Y444+500+730F+T492V AAV2 quadruple-mutant (QM) capsid (WT-Fluc-AAV2/QM); (ii) two GenX genomes and AAV2 QM capsid (LC1-Fluc-AAV2/QM and LC2-Fluc-AAV2/QM); (iii) WT genome and S663V+T492V AAV3 double-mutant (DM) capsid (WT-Fluc-AAV3/DM); and (iv) two GenX genomes and AAV3 DM capsid (LC1-Fluc-AAV3/DM and LC2-Fluc-AAV3/DM). The combination of the modified-genomes with the capsid-mutants led to ~5-6-fold increase with both AAV2 and AAV3 serotype vectors following transduction of a human hepatocellular carcinoma (HCC) cell line, Huh7, at an MOI of 1,000 vgs/cell under identical conditions in vitro. When male C57BL/6 mice were injected via tail-vein with WT-Fluc-AAV2/QM, LC1-Fluc-AAV2/QM, and LC2-Fluc-AAV2/QM vectors at a relatively low dose of 5×109 vgs/mouse, the AAV2 QM capsid-mutant vectors led to ~6-10-fold increase in transgene expression in the liver. Similarly, when WT-Fluc-AAV3/DM, LC1-Fluc-AAV3/DM, and LC2-Fluc-AAV3/DM vectors were injected via tail-vein in NSG mice xenografted with human liver tumors at relatively low dose of 3×109 vgs/mouse, led to ~5-8-fold increase with the combination of modified-genomes with the AAV3 DM capsid-mutant vectors, which was restricted to human liver tumors. Taken together, these data document that the combination of NextGen capsids and GenX genomes leads to the generation of optimized [Opt] AAV serotype vectors (Fig. 1CFig. 1C), which transduce cells and tissues more efficiently, both in vitro and in vivo, at significantly reduced doses. These studies have significant implications in the potential use of the Opt AAV serotype vectors in human gene therapy.View Large Image | Download PowerPoint Slide

160. Successful Treatment of Neonatal Metachromatic Leukodystrophy Model Mice by Low Dose of Self-Complementary AAV Type9 Vector Expressing ASA

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease (LSD) caused by a functional deficiency of the lysosomal enzyme arylsulfatase A (ASA) and characterized by severe neurological symptoms due to widespread and progressive demyelination in the both central and peripheral nervous systems. Enzyme replacement therapy (ERT) has been applied to treat certain types of LSDs, but correction of neurological abnormalities is usually hampered by the blood brain barrier (BBB). We have previously shown that MLD mice can be treated by intravenous (IV) injection of classical single-stranded AAV type 9 vector expressing ASA (ssAAV9/ASA) for neonate and self-complementary AAV9/ASA (scAAV9/ASA) for adult. To develop the clinical MLD gene therapy, it is important to decrease the amount of viral vectors because of safety. In this study, we evaluated the therapeutic potential of scAAV9/ ASA-mediated systemic transduction of neonatal MLD mice at 10 times lower dose (2 × 1011 v.g./body) compared to the treatable dose of the ssAAV9/ASA (2 × 1012 v.g./body). We generated both ssAAV9/ASA and scAAV9/ASA to intravenously inject then into the neonatal MLD mice (n=6-9). ELISA analysis showed that sustained high-level expression of ASA was detected in the brain and spinal cord of the scAAV9/ASA-treated mice (cerebral cortex, 2.1 times; cerebellum, 2 times; spinal cord, 5.5 times) compared to the ssAAV9/ASA for more than one year. Furthermore, in the behavior test, both scAAV9/ASA and ssAAV9/ASA-treated mice showed a significant improvement in their ability to traverse narrow balance beams, as compared to the non-treated MLD mice (latency; 7.2±0.9, 9.3±1.1 vs. 16.6±0.8 sec, P<0.05: slips; 3.4±0.8, 3.6±0.6 vs. 7.3±1.8 times, P<0.05). These data clearly demonstrate that MLD model mice can be treated by systemic neonatal transduction with low dose of scAAV9/ASA. Therefore, neonatal gene therapy would be an important option for parents faced with the prenatal diagnosis of a genetically affected child. Since ten times lower dose of scAAV9/ASA is enough to treat the neonatal MLD mice compared to ssAAV9/ASA, transduction strategy to use scAAV9/ASA should be safe and more practical for the clinical MLD gene therapy.

677. Stabilizing the rAAV Vector Genome Increases AAV Vector Transduction Efficiency

It has been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. Administering rAAV vectors in conjunction with a drug that can enhance vector transduction may overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Here we discovered that Cidofovir(CDV), a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase, is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 9 fold, depending on the cell type and concentration. This increase has been consistently observed in 16095, HelaS3, and Hek293 cells with little toxicity to the cells. However, there is less than a two-fold increase with Cidofovir treatment when using self-complementary AAV vectors(scAAV), suggesting that CDV might enhance conversion of the AAV genome from single to double stranded form. These observations were confirmed using two different serotypes, AAV2 and AAV8, in vitro indicating that the mechanism of enhancement is not serotype dependent. Southern Blot analysis along with real-time PCR demonstrated that Cidofovir treatment in HelaS3 cells increases viral DNA accumulation 24 hours post-infection compared to PBS treated cells. Cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hrs post infection, suggesting CDV does not enhance viral entry. However, cytoplasmic and nuclear fractions of HelaS3 cells pretreated with CDV or PBS demonstrated that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection.In vivo, Hemophilia A BALB/c(BALB/c-HA) mice were treated with CDV prior to infection with a rAAV containing the fVIII Human Light chain(LC) gene. It showed that CDV treatment enhanced the amount of LC antigen when compared to control group. Approximately two fold increase was observed up to fourteen weeks post-injection. These results demonstrate that CDV treatment can enhance and sustain rAAV levels in vivo.CDV has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, have not been documented. The use of drugs like Cidofovir can greatly contribute to the understanding of rAAV trafficking and eventually lead the development of novel strategies to increase overall efficiency of AAV transduction.

676. DNA Sequences Encoding shRNAs Can Replace Mutant ITR in scAAV Genome for Efficient Replication and Packaging and Transcribe shRNAs by pol III Promoter Activity of wt ITR for Efficient Gene Silencing

The two inverted terminal repeats (ITRs) at each end of recombinant adeno-associated virus (rAAV) genome are the only sequences inherited from wild type (Wt) viral genome. They contribute to rAAV genome replication, packaging, intracellular processing and stabilization, and integration into host genome. Previously we showed DNA sequences encoding shRNAs (shDNA) are the barriers in self-complementary AAV (scAAV) genome replication, resulting in vector genome truncations and heterogeneity. We now observe the same phenomena in the genome replication and packaging of single-stranded AAV vectors harboring shDNA. When detected on a denaturing gel, these truncated genomes were doubled in size as seen for scAAV, indicating they are intra-molecular double-stranded DNA molecules. We hypothesized DNA with hairpin structures could serve as the mutant ITR during rAAV genome replication and packaging. To test this, we replaced the mutant ITR in scAAV genome with the DNA encoding shRNA against Apob or Firefly luciferase gene. Southern blot of Hirt's DNAs revealed that vector genomes flanked with shDNA and wtITR, but not with shDNA and mutant ITR, were efficiently rescued and replicated when co-transfected with adeno-helper plasmid and Rep/Cap trans-plasmid into HEK293 cells. Large scale AAV production and analyses of CsCl gradient purified rAAV genomes in both native and denaturing gels further demonstrated that hybrid shDNA-wtITR vector genomes were efficiently packaged into AAV capsid as double-stranded genomes. To test their functionalities in vivo, we intravenously injected 2×1011 GCs of scAAV9 containing shApoB-wtITR genome with an EGFP gene into adult C57 mice and harvested liver tissues 3 weeks later. Compared to regular scAAVEGFP vector, the shApoB-wtITR AAV vectors achieved comparable EGFP transduction efficacy in the liver. Interestingly, without a H1 promoter the shApob produced abundant small antisense RNA and silenced mouse ApoB gene as efficienct as conventional scAAVshRNA vectors. Southern blots of mouse liver detected linear and circularized forms of AAV genomes, thus implying that wtITR in the circularized genome may serve as a strong pol III promoter to drive shRNA expression. Currently, we are in the process of replacing Wt ITR with an artificial chimeric ITR with only rep binding element, terminal resolution sites and D sequence of authentic ITR retained. In summary, hairpin structure sequences can function as an alternative mutant ITR for production of functional scAAV. Our findings may have significant impact on AAV biology and vectorology, potentially enabling us to create artificially intelligent and programmable vector genomes with improved packaging efficacy, expanded genome sizes, modulated tissue tropism and propensity of integration, regulatable in vivo persistence and potency, etc.

101. Generation of Improved Recombinant AAV3 Serotype Vectors Using AAV3 ITRS and AAV3 REP Proteins

Although recombinant AAV3 serotype vectors were largely ignored previously, owing to their poor transduction efficiency in all cells and tissues examined, our initial observation of their selective tropism for human liver cancer cell lines and primary human hepatocytes (Mol Genet Metabol., 98: 289-299, 2009; Hum Gene Ther., 21: 1741-1747, 2010; Gene Ther., 19: 375-84, 2012), has led to renewed interest in this serotype, since AAV3 vectors and their variants have now proven to be extremely efficient in targeting human and non-human primate hepatocytes in vitro as well as in vivo (Mol Ther., 22: S2, 2014; Mol Ther., 22: S91, 2014; Nature, 506: 382-386, 2014; Hum Gene Ther., 25: 1023-1034, 2014). Previously, it wasreported that the combination of ITR8 with AAV8 capsids (AAV8/8) resulted in vectors that led to at least 2-fold increase in transgene expression in mouse liver, compared with AAV8 capsids pseudotyped with ITR2 (AAV2/8) vectors (Mol. Ther. 11: S156, 2005), and that the ITRs from AAV serotypes 1-6 were interchangeable when they are packaged into AAV8 capsids (AAV1/8 – AAV6/8), but played no role in transgene expression in murine hepatocytes in vivo (J. Virol., 80: 426-439, 2006). In our present studies, we wished to evaluate the relative contribution of the cis-acting ITR from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the recombinant AAV3 vector production and transduction. To this end, we utilized two helper plasmid: pAAVr2c3, which carries rep2 and cap3 genes and pAAVr3c3, which carries rep3 and cap3 genes. Plasmid transfection assays revealed that both AAV2 and AAV3 Rep proteins were expressed at similar levels in the presence of adenoviral helper genes. We also generated two sets of single-stranded AAV vector constructs carrying an expression cassette containing the EGFP reporter gene flanked by either ITR2 or ITR3. Plasmid transfections of the ITR2- or the ITR3-containing expression cassettes into cultured cells also revealed no difference in the extent of transgene expression. The combination of ITR3 and Rep3 resulted in ~5-fold higher rAAV3 vector titers, as determined by quantitative PCR assays. Next, purified viral stocks of AAV3 vectors were generated and designated as Rep2-ITR2 and Rep3-ITR3, respectively. Interestingly, the transduction efficiency of Rep3-ITR3 AAV3 vectors was ~4-fold higher than that of Rep2-ITR2 AAV3 vectors in a human hepatocellular carcinoma cell line, Huh7, under identical conditions. Additional studies are currently underway to evaluate the efficacy of the Rep2-ITR2 and Rep3-ITR3 vectors in a murine xenograft model in vivo. In summary, our studies document that the combined use of the AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids lead to the production of recombinant AAV3 vectors with higher titers and with higher transduction efficiency, and suggest that the use of homologous ITRs, Rep proteins, and capsids are similarly likely to be applicable to other AAV serotypes vectors for their optimal use in human gene therapy.

Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

BackgroundSelf-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX).MethodsHemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8+ T cell responses to hF.IX.ResultsSelf-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8+ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome.ConclusionsThe nature of the AAV genome can impact the CD8+ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.

Double-strand adeno-associated virus-mediated exendin-4 expression in salivary glands is efficient in a diabetic rat model

AimExendin-4 (Ex-4) is an agonist of the glucagon-like peptide 1 (GLP-1) receptor, approved for the treatment of type 2 diabetes (T2DM). Several strategies have been tried to develop stable and efficacious Ex-4 expression systems. The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of exendin-4 in salivary glands (SG), improves pathology in the Sprague–Dawley (SD) rat model of diabetes mellitus (DM). MethodsThe effects of Ex-4 expression by recombinant dsAAV-NT4-Ex-4 were evaluated in vitro compared with a single-strand (ss) AAV. The dsAAV was delivered into SGs and the blood glucose and insulin levels were assessed in a rat model of DM. ResultsDsAAV-NT4-Ex-4 virus induces significant exendin-4 expression in vitro. Furthermore, Ex-4 expressed from dsAAV virus in SGs enhances insulins secretion in vivo and significantly controls the onset of hyperglycemia in rat model of DM. ConclusionsResults suggest that sustained secretion of Ex-4 following dsAAV-mediated gene therapy is feasible. SGs appear to be promising targets with potential clinical applicability for the treatment of DM. This represents the example of a successful use of Ex-4 for diabetes therapy, providing support for direct AAV-mediated in vivo as an easy, safe and efficient therapeutic strategy.

Self-Complementary AAV Vectors Delivered Intramuscularly Do Not Break Tolerance In a Partial Knockout Model Of Hemophilia B

Self-complementary adeno-associated viral (scAAV) vectors have become a popular tool for AAV gene therapies due to their increased transgene expression relative to single-stranded (ss)AAV vectors, at least for models in which the transgene can fit within these restrictive constructs. The most recent clinical trial for hemophilia B used a scAAV8 vector. However, recent reports by our lab and others have suggested that these vectors are also more immunogenic than their single-stranded brethren. We previously showed that, during hepatic gene transfer, the innate immune response is greater with a scAAV vector. Moreover, Wu et al. demonstrated an enhanced T and B cell response to a secreted form of HIV's gag protein when it was delivered via scAAV. While this was beneficial for their vaccine model, it could potentially be problematic for therapeutic gene transfer applications. To investigate whether this phenomenon could deleteriously impact gene therapy for hemophilia B, we used a partial knockout model of hemophilia B. This mouse model has the murine factor IX gene knocked out, and then a truncated form of human factor IX (hF.IX) is inserted. Specifically, the mice we used have a late stop codon (LS) at amino acid 338; while hF.IX can be detected in the liver, this mutated protein is not released into circulation (crm-). During muscle gene transfer with AAV2, LS mice have an interesting response: while they form antibodies to hF.IX, there is no CD8+ T cell infiltrate detected in the transduced tissue. We then set out to determine whether this partial tolerance could be disrupted using a scAAV vector. Using AAV1, which is a more relevant serotype for muscle gene therapy, we injected single-stranded and self-complementary vectors (1011 vg/mouse) and measured the resulting immune responses. Surprisingly, unlike with AAV2, the LS mice completely tolerated AAV1-mediated intramuscular delivery of hF.IX. They did not make detectable anti-hF.IX IgG1, and their plasma had no Bethesda activity through 4 weeks post-injection. In contrast, hF.IX null mice (HBKO) made 10708±3869 ng anti-hF.IX IgG1 and developed inhibitory activity of 22.5±26 Bethesda units 4 weeks after delivery of a scAAV vector. Consequently, LS mice had circulating hF.IX levels of 148.9±18.64 (ssAAV1) and 60.12±8.71 ng/mL (scAAV1), while hF.IX was undetectable in the plasma of HBKO mice at this time point. The CD8 response was similarly lacking in LS mice, with splenic responses to hF.IX detected by ELISPOT reduced by ∼2-10 fold relative to HBKO mice. Infiltrating CD8 cells were detected in the muscle of HBKO but not LS mice. In conclusion, our data suggest that the underlying mutation within the patient is a more important risk factor for immune responses to the transgene than the molecular form of the AAV genome. Disclosures:Herzog:Genzyme: AAV technology, AAV technology Patents & Royalties.

No tumour-initiating risk associated with scAAV transduction in newborn rat liver

Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.

Adeno-associated viral vector-mediated transgene expression is independent of DNA methylation in primate liver and skeletal muscle.

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.

Preclinical Differences of Intravascular AAV9 Delivery to Neurons and Glia: A Comparative Study of Adult Mice and Nonhuman Primates

Other labs have previously reported the ability of adeno-associated virus serotype 9 (AAV9) to cross the blood-brain barrier (BBB). In this report, we carefully characterized variables that might affect AAV9's efficiency for central nervous system (CNS) transduction in adult mice, including dose, vehicle composition, mannitol coadministration, and use of single-stranded versus self-complementary AAV. We report that AAV9 is able to transduce approximately twice as many neurons as astrocytes across the entire extent of the adult rodent CNS at doses of 1.25 × 10¹², 1 × 10¹³, and 8 × 10¹³ vg/kg. Vehicle composition or mannitol coadministration had only modest effects on CNS transduction, suggesting AAV9 crosses the BBB by an active transport mechanism. Self-complementary vectors were greater than tenfold more efficient than single-stranded vectors. When this approach was applied to juvenile nonhuman primates (NHPs) at the middle dose (9-9.5 × 10¹² vg/kg) tested in mice, a reduction in peripheral organ and brain transduction was observed compared to mice, along with a clear shift toward mostly glial transduction. Moreover, the presence of low levels of pre-existing neutralizing antibodies (NAbs) mostly occluded CNS and peripheral transduction using this delivery approach. Our results indicate that high peripheral tropism, limited neuronal transduction in NHPs, and pre-existing NAbs represent significant barriers to human translation of intravascular AAV9 delivery.

131. A Simple Strategy To Increase the Transduction Efficiency of Single-Stranded AAV Vectors In Vitro and In Vivo

131. A Simple Strategy To Increase the Transduction Efficiency of Single-Stranded AAV Vectors In Vitro and In Vivo

Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors

Central nervous system (CNS) disorders are important targets for gene therapy; however, delivery of therapeutic proteins and/or genes to the brain remains a major challenge due to the difficulty of efficiently delivering viral vectors across the blood–brain barrier (BBB). In the present work, we tested the ability of several single-stranded adeno-associated viral (ssAAV) serotypes to deliver transgenes to the brain and spinal cord in neonatal mice. We injected ssAAV vectors encoding GFP (serotype-1, -8, -9 and -10: 1.5 × 10 11 vector genomes each) into the jugular vein of neonatal mice and assessed GFP expression immunohistochemically. Strong GFP signals were detected in both the brain and spinal cord after injection of any of these serotypes. ssAAV serotype-9 mediated gene transfer was the most efficient. GFP expression was detected throughout the brain, including the cortex, cerebellum, olfactory bulb and brainstem and was sustained for at least 18 months. Immunohistochemical staining showed that the GFP signals were detected in GFAP positive astrocytes, NeuN positive neurons, and Calbindin positive purkinje cells. Our data suggest that systemic neonatal injection of ssAAV is an effective strategy for delivering transgenes to target neuronal systems that are not accessible to viral vectors in adult animals. These vectors should prove highly useful for efficient and long-term overexpression or downregulation of genes in CNS and spinal cord and could be a useful means of treating genetic neurological diseases.